Vaccination is the most important public health measure for preventing disease caused by viral infection. The effective use of vaccines is dependent on being able to quickly produce large quantities of vaccine material (e.g., virus) from a stable and easy to cultivate source. The rapid development of vaccines and their abundant availability is critical in combating many human and animal diseases. Delays in producing vaccines and shortfalls in their quantity can cause problems in addressing outbreaks of disease. For example, recent studies suggest that there is cause for concern regarding the long lead times required to produce vaccines against pandemic influenza. See, for example, Wood, J. M., 2001, Philos. Trans. R. Soc. Lond. B. Biol. Sci., 356:1953. Accordingly, recent efforts to produce vaccines have focused on growth of viruses for vaccines in cell culture.
3.1 Influenza Virus
In particular, the use of Madin Darby Canine Kidney (MDCK) cells has been pursued by a number of groups. See, for example, U.S. Pat. No. 6,455,298; U.S. 2005/0118140; U.S. 2005/0118698; U.S. Pat. No. 6,825,306; WO2005/113758; and Radaeva, I. F., et al. Vopr. Virusol. (2005) 50: 43-6. However, many of the existing MDCK cell lines suffer from one or more defects including tumorigenicity, the requirement for animal serum in cell culture, and low yields of influenza viruses suitable for use in vaccines. Furthermore, many of the cell culture processes which have been developed for the production of vaccine material from these cell lines often require numerous manipulations including medium exchange steps and the use of large amounts of virus for inoculation. In addition, because of the continual emergence (or re-emergence) of different influenza strains, new influenza vaccines are generated each season based on the circulating influenza strains. Unfortunately, some influenza vaccine strains are more difficult to grow to high yields. The yield of each batch of cell culture derived material not only defines production capacity but also impacts the cost of manufacturing product thus improving viral yield (i.e., peak viral titer) is desirable.
Recently, novel serum-free medium and non-tumorigenic cell lines that can grow vaccine strains to very high titers and processes for the production of viral material in disposable bioreactors have been developed (see, U.S. 2006/0188977; and U.S. Ser. No. 11/855,769 filed Sep. 14, 2007). The instant invention expands this work and provides novel cell culture medium, highly reproducible efficient scalable processes for the production of large quantities of vaccine material in bioreactors from MDCK cells, in particular, non-tumorigenic cells lines, without the need for medium exchange, and robust downstream purification processes for the production of a highly purified live attenuated vaccine. The methods provided by the present invention are robust requiring minimal manipulation and are cost effective.
3.2 Respiratory Syncitial Virus (RSV)
Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection (LRTI) in infants and young children and is responsible for considerable morbidity and mortality. A total of 18 million people are infected annually in the seven major markets (US, Japan, France, Germany, Italy, Spain, UK), including three million adults and almost 400,000 premature infants. Annually in the U.S. alone, it is estimated that 70,000-125,000 hospitalizations are attributed to RSV LRTI. Two antigenically diverse RSV subgroups A and B are present in human populations. RSV is also recognized as an important agent of infection in immuno-compromised adults and in the elderly. Due to the incomplete resistance to RSV reinfection induced by natural infection, RSV may infect multiple times during childhood and life. The goal of RSV immunoprophylaxis is to induce sufficient resistance to prevent the serious disease which may be associated with RSV infection. The current strategies for developing RSV vaccines principally revolve around the administration of purified viral antigen or the development of live attenuated RSV for intranasal administration. However, to date there have been no approved vaccines or for RSV.
The viral genomic RNA is not infectious as naked RNA. The RNA genome of RSV is tightly encapsidated with the major nucleocapsid (N) protein and is associated with the phosphoprotein (P) and the large (L) polymerase subunit. These proteins form the nucleoprotein core, which is recognized as the minimum unit of infectivity (Brown et al., 1967, J. Virol. 1: 368-373).
Despite decades of research, no commercially available safe and effective RSV vaccine has been developed for the prevention of severe morbidity and mortality associated with RSV infection. A formalin-inactivated virus vaccine has failed to provide protection against RSV infection and in fact lead to exacerbated symptoms during subsequent infection by the wild-type virus in infants (Kapikian et al., 1969, Am. J. Epidemiol. 89:405-21; Chin et al., 1969, Am. J. Epidemiol. 89:449-63). Efforts since have focused on developing live attenuated mutants obtained by recombinant methods, chemical mutagensis and cold passage of the wild type RSV for temperature-sensitive mutants (Gharpure et al., 1969, J. Virol. 3: 414-21; Crowe et al., 1994, Vaccine 12: 691-9). However, purification of live attenuated virus from their cell-associated proteins, which would meet regulatory guidelines for efficacious prophylactic treatment of RSV infection, has proved elusive until now.
Similar to the influenza virus, RSV is grown in a stable cell line wherein the virus is closely associated with the host cell making it difficult to purify the virus from the cell-associated proteins while maintaining a minimum unit of infectivity. Adding to the complexity, RSV is fragile (e.g., shear sensitive). These factors, the virus being predominantly host cell associated and fragile, have made it difficult to purify the virus from host cell extract (e.g., DNA and proteins) while resulting in a clinically acceptable immunogenic composition comprising live attenuated virus. It is for these reasons, in part, why no vaccines are commercially available for RSV. Accordingly, there is a need for a purification process that can be used to purify RSV and other cell-associated viruses for preparation and formulation of immunogenic compositions.